ANALYTICAL CHEMISTRY
174
rate of hydrolysis (measured by the slope of the line) and extrapolating to zero rate, as shown in Figure 4. For the authors' particular sample, the extrapolated value corresponds exactly t o the hydroxydilaurate, showing that this was the soap originally present. It appears therefore that the effect of hydrolysis can be accounted for completely only by a double extrapolation t o zero time and zero rate of hydrolysis.
5
1.0
-
A
A L W N Y M LAUPATE W X T W T E D *sii.,aSXnipl
0 A I I N I W M STEMATE
P.WNEXTWTED * s H . S . a 7 X A L A )
EXPERIMENTAL
--- AL&IN*I
2.8'-
3.0*
I
1
I
D ( w
I
I
SLOPE, A S H , %/LITER
0 , 2 3 1 5 The aluminum laurate was prepared by addition of potassium laurate solution to excess aluminum A C E T O N E , LITERS/GRAM OF S O A P Figure 4. Variation of Comnitrate solution a t the boiling point, as previously position of Original Soap (Obdescribed (4. Soaps Precipitated a t lower ternFigure 3. Effect of Fatty Acid upon tained by Extrapolation of Peratures showed more rapid and more extensive Composition of Aluminum Soaps Flat Portion of Curves of hydrolysis. Soxhlet extractors modified ( 4 )for conSoxhlet-Extracted under Least Hy- Figures 1 and 2) with Rate of trol of throughput and temperature were used, and drolyzing Conditions (2" C., Acetone Hydrolysis (Slope of These t h e total volume of liquid which refluxed through Portions) Dried over Drierite) t h e sample was computed. The acetone was dried several days over the drying agent and a fresh portion of the same was placed in the boiling flask. Unless other(3) RIysels, K. J., J . Phys. & Colloid Chem., 51, 708 (1947). wise indicated, Drierite (anhydrous calcium sulfate) was used. (4) Mysels, K. J., Pomeroy, H. H., and Smith, G. H., ANAL.CHEM.. T h e soaps were dried over phosphorus pentoxide in vacuum for 20, 878 (1948). several days. The composition of the soap after extraction was (5) Smith, G. H., pomeroy, H. H., MoGee, C. G., and Mysels, K. J., determined by ashing. The ash was completely insoluble in J . Am. Chem. Soc., 70,1053 (1948). water and was reported as aluminum oxide (AlzOa). Each point (6) Timmermans, J., a n d Gillo, L., Rocnilci Chem., 18, 812 (1938). in the curves represents a separate extraction beginning Kith a fresh sample of the same dried soap.
LITERATURE CITED
(1) Back, E., and Aimin, K. E., Acta Chem. Scand., 4, 1401 (1950). (2) Coe, R. H., Mysels, K. J., and Smith, G. H., J . Colloid Sei., 3, 293 (1948).
RECEIVED for review July 16, 1952. Accepted September 9, 1952. This study was conducted under Contract OEMSR-1057 between Stanford University and the O 5 c e of Emergency htanagement recommended b y Division 11.3 of the National Defense Research Council, and supervised by ,J. W . McBain.
Precision Obtained with Vibrating Reed Electrometer in Radioassaving Meta- and Para=Substitutei Benzoic-Alpha-Carbon-14 Acids I
I
VERNOK F. RAAEN AND GUS A. ROPP Chemistry Dicision, Oak Ridge National Laboratory, Carbide & Carbon Chemicals Co., Oak Ridge, Tenn. URING a quantitative study of the isotope effects in the alkaline hydrolysis of a series of meta- and para-substituted ethyl benzoates labeled with carbon-14 in the carboxyl group, it was necessary to obtain precise values of the specific activities of a number of qamples of the tracer-labeled acids. A description of this study is published elseahere ( 2 ) . The specific activities of the acids reported were used in calculating the isotope effects on the rates of hydrolysis of the various esters. The data gathered also demonstrated the degree of precision obtainable in the over-all process of 17an Plvlce combustion of a measured sample ( I ) , sweeping of thc carbon-14 dioxide into an ion chamber (I), and assaying ralioactivity ( 1 ) . The errors normally resulting from weighing the micro samples were almost entirely eliminated by accurately aliquoting the benzoic acid samples in solution. PREPARATION OF SUBSTlTUTED BENZOIC ACID SA3II'LES
Fifty millimoles of carefully purified, acid-free ethyl benzoatr-acarbon-14 were dissolved in 100 ml. of absolute ethyl alcohol and brought to the temperature of a thermostat, 25' C., in a closed vessel. Enough 0.15 h- sodium hydroxide was added from a pipet t o hydrolyze 3% of the ester. When the reaction was complete as indicated by fading of the color of added phenolphthalein, the excess ester was quickly extracted from the solution with ether. The benzoic acid released was recovered b r acidification and ether extraction. The acid sample was carefully purified by recrystallization from water, followed by vacuum sublimation. I n other experiments, enough 0.30 N sodium hydroxide was added t o effect 6% hydrolysis of the ester. For 100% hydrolysis of the ester a large excess of sodium hydroxide was used, I n all cases the purification of the acids involved recrystallization from n ater followed by a single vacuum sub-
limation. limation.
Certain samples were treated t o one additional subWET COMBUSTION OF ACID SAMPLES
Six to nine samples of each acid were assayed. To obtain samples approximately 80 mg. of acid was weighed carefully, dissolved in dilute sodium hydroxide solution, and made up to 50 ml. in a volumetric flask. Five-milliliter aliquots were pipetted into short 12-ml. glass test tubes with 19/38 male joints which fit the wet combustion apparatus. The water waso removed from the aliquots by evaporation in an oven a t 70 C. The solids remaining were subjected to a e t combustion as previously described (1). For the combustion of p-chlorobenzoic acid samples, a slight modification of the usual wet combustion procedure was used to prevent reaction of the free chlorine released with the mercury in contact with the frit valve. The difficulties previously encountered in the wet combustion of halogen compounds were rompletely eliminated. The modification consisted of placing in the apparatus a U-tube filled with Stannous chloride crystals through which the carbon dioxide released from the oxidizing solution passed on its way t o the frit valve ( 1 ) and ion chamber. Passage of the gases through the stannous chloride appeared to remove chlorine quantitatively. RADIOASSAY OF SAMPLES
The carbon-14 dioxide from combustion of each aliquot was swept with inactive carbon dioxide into a 250-ml. Borokowsky stainless steel ionization chamber (1 ). Radioassays were performed using the Applied Physics Corp. 450-cycle vibrating reed electrometer (Model 30), which was kept in a constant temperature room. The effective input capacitance including the ionization chamber was approximately 17.2 ppf. The "rate of drift" method or rate of charging of a condenser was used EO that readings were recorded in seconds required to charge a specified con-
i 75
V O L U M E 2 5 , NO. 1, J A N U A R Y 1 9 5 3 Table 1. -4ssay Data
Acid from
5‘% of Ester
Hydrolyzed
102 X Specific Activity,
Mean
“95Yo Interval Confidence of Mean” Interva! in yo of of Mean ’ Mean
rc./Mg.
Ethyl benzoate-acarbon-14 Ethyl-p-chlorobenaoate. u-carbon-14
100 3 100
3
6 Ethyl-p-toluate-acarbon-14
100
3 6
Ethyl-m-nitrobenzoatear-carbon-14 a
100
8.22, 8.20,8.22, 8.18, 8.21, 8.208 8.22 7.58, 7.59, 7.63, 7.63. 7.64, 7.615 7.62 7.00, 7.02, 7.00, 7.04, 7.03, 7.024 7.05, 7.03 6.47, 6.49, 6.48, 6.48, 6.47, 6.480 6.47, 6.50 6.49, 6.48, 6.47, 6.52, 6.47, 6.488 6.50 1.999, 1.996, 2.006, 2.006. 2.0060 2.010,2.012, 2.013, 2.006 1.872, 1.853, 1.865, 1.861, 1.8645 1.863,1.870.1.865, 1.867 1.845. 1.877, 1.852. 1.863, 1,8606 1.850,1.873,1.861.1.857, 1.868 1,530, 1.530, 1.545, 1.535. 1.5396 1.520,1.545,1.550,1.550,
10.013
=kO 16
f0.020
ztO.26
10.014
z t o , 20
10.009
10.14
iO.015
i0.23
&0.004
%0.20
10.002
i o , 22
10.007
10.38
%0.007
‘0.45
coYcLusIos
1 R.51
l.j20;-1.523, 1.530, 1.535. 1.5340 ztO.008 io.52 1.540. 1.545. 1.545 1.446, 1.432, 1.427, 1.428, 1.4399 3 =to.007 ztO.49 1.438, 1.448, 1.452. 1.448 Ethyl-p-aninate-rr1.414, 1.414, 1.414, 1.415, 1.4128 100 i.o.002 10.14 carbon-14 1.410, 1.410, 1.406, 1.412, 1.417 3 1.280, 1.276, 1.295, 1.295, 1.2926 10.007 50.54 1.305. 1.293, 1.295, 1.302 1.297, 1.303. 1.300, 1.300. 1.3010 6 i0.002 i.o.15 1.297 1.307, 1.300. 1.303, 1.302 Average “95% confidence interval of nieaii” = 1 0 . 2 9 % , a Prepared b y resublimation of sample of acid from 100‘7, hydrolysis of ethyl-m-nitmbenaoaterr-rarbon-14. loon
The corrected mean rate of drift values was multiplied by a factor to convert to the specific activity values listed in column three of Table I. The six to nine specific activity values n-ere averaged to obtain the mean values sholyn in CYJlUlllll four. Columns five and six list t,he “95% confidence intervals of the means” of the specific activities for the various acid samples assayed. Each of these confidence intervals was r:ilrulated as twire t,he “standard deviation of the mean.” The average of all the values of the confidence intervals in percentages (column six) is also indicated a t the bottom of the table.
Under thr conditions used in this work the average precision of the overall proceps of Van Slyke conibustion and radioassaving of the various carboxvl-labeled benzoic acids was mean ~ k 0 . 3 7for ~ the approximate 95”i, confidence interval of the mean of 6s t o nine assays. To obtain such precision it was necessary to perform all the assays on a given .ample of acid in one day. ACKNOWLEDG\IE\T
drnser to a given potential. For each 5-mL aliquot thc mran of ten observed “rates of drift” WHS recorded. T o correct for variations in the instruments, a series of “rates of drift” of a standard uranium chamber was recorded immediately after measuring each serie? of rates of drift resulting from a single wet combustion of a 5-ml. aliquot. The standard uranium chamber consisted of H 250-ml. gold-plated brass chamher containing a piece of uranium wrapped Kith aluminum to absorb alpha particles. Standardized against sodium carbonatecarbon-14 solution supplied by the Sational Bureau of Standards, it had a value equivalent to 0.1015 pc. of carbon-14. Mean ratee of drift readings were corrected to this stantlard by multiplication by a factor as follows: Correrkd mean rate of drift for 5-ml. aliquot = mean rate of drift from observed values X calculated rate of drift of standard mean rate of drift of standard from observed values
The modification to the xvet comhustion piocedure, used to prevent reaction of free chlorine I\ ith the mercury in contact with the frit valve, was developed by H. TI-, Davis of the University of South Carolina. 0. K. Neville and A. J . Weinberger prepared the standard used t o corrert Eo1 variation? in the instruments. LITERATURE CITED
(1) Xeville, 0 . K., J . Am. Chem. SOL..70, 3501 11948).
(2) Ropp, G. A , , and Raaen, V. F., J . Chetn. Phys. in press RECEIVED for review June 0, 1052. Accepted September 26, 10.52. Based o n work performed under contract W-7405-eng-26 for the I t o m i c Enern? Commission a t Oak Ridge National Laboratory.
Quantitative Estimation of the Carbonyl Groups in Saturated Ketocholanic Acids E. L. P R A T T , Winthrop-Steurns Inc., Rensseluer, ,V. Y.
S E of the accepted proceclurc~sused in large scalp conversion of cholic acid (3,7,12-trihydroq-rholanic acid) to dmowcholic arid (3,12-dih~droxycholanicarid) requires a selective oxidation of the 7-hydroxy group a i t h bromine to a monoketocholnnic arid. T h c carbonrl group is then rliminatcd through a WolffKishner rcduction. This paper proposes an analytical method for rapid control testing in the above process. The techniques described can be extended to the evaluation of certain saturated di- and triketorholanic acids. GENERAL
The literature contains various methods for estimating carbonyl compounds. Generally, these are procedures based on gravimetric assay of reaction products such as hydrazones ( d ) , semicarbazones (3), oximes ( 5 ) , hydantoins ( I ) , etc. The estimation of free acid after the oxime formation with hvdroxyl-
amine hydrochloride has been reported ( 6 ) . Very excellent polarographic techniques have been published, but these generally necessit,ite preliminary derivative prepzrcttion ( 7 , 8 ) . Little or nobhing has been repoi ted on the spc’ctrophotonietric estimation of ketones in the saturatcd rholanic acid series. T e t this approach presents a procedure \\hi( h is eltreniely rapid and. of even greater consequence, it is a nirLtwie of the kcto unit as such and not the measure of a rcaction product I n general, saturated slstenis do not ‘Lbsorb ultraviolet light and their spectra are studied in thr loxr frequency or infrared region. (In this discussion thr t e x t ni ultraviolet actually refers to that region of the ultraviolet spectrum which is easily studied and is limited to those wave lengths exceeding 220 nip.) Desoxycholic arid is an example of a saturated system in the bile acid series which shows no absorption in the ultraviolet region of the spectrum. If, however, a saturated cholanic acid possesses one or more keto groups, the carbonyl resonanre can br detected
